Am J Physiol Cell Physiol 307: C928–C938, 2014. First published August 27, 2014; doi:10.1152/ajpcell.00244.2014.

Mechanisms of NFATc3 activation by increased superoxide and reduced hydrogen peroxide in pulmonary arterial smooth muscle Juan Manuel Ramiro-Diaz,1 Wieslawa Giermakowska,1 John M. Weaver,2,3 Nikki L. Jernigan,1 and Laura V. Gonzalez Bosc1 1

Vascular Physiology Group, Department of Cell Biology and Physiology, School of Medicine, University of New Mexico Health Sciences Center, Albuquerque, New Mexico; 2Center of Biomedical Research Excellence, University of New Mexico Health Sciences Center, Albuquerque, New Mexico; and 3Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, New Mexico Submitted 11 July 2014; accepted in final form 25 August 2014

Ramiro-Diaz JM, Giermakowska W, Weaver JM, Jernigan NL, Gonzalez Bosc LV. Mechanisms of NFATc3 activation by increased superoxide and reduced hydrogen peroxide in pulmonary arterial smooth muscle. Am J Physiol Cell Physiol 307: C928–C938, 2014. First published August 27, 2014; doi:10.1152/ajpcell.00244.2014.—We recently demonstrated increased superoxide (O2·⫺) and decreased H2O2 levels in pulmonary arteries of chronic hypoxia-exposed wild-type and normoxic superoxide dismutase 1 (SOD1) knockout mice. We also showed that this reciprocal change in O2·⫺ and H2O2 is associated with elevated activity of nuclear factor of activated T cells isoform c3 (NFATc3) in pulmonary arterial smooth muscle cells (PASMC). This suggests that an imbalance in reactive oxygen species levels is required for NFATc3 activation. However, how such imbalance activates NFATc3 is unknown. This study evaluated the importance of O2·⫺ and H2O2 in the regulation of NFATc3 activity. We tested the hypothesis that an increase in O2·⫺ enhances actin cytoskeleton dynamics and a decrease in H2O2 enhances intracellular Ca2⫹ concentration, contributing to NFATc3 nuclear import and activation in PASMC. We demonstrate that, in PASMC, endothelin-1 increases O2·⫺ while decreasing H2O2 production through the decrease in SOD1 activity without affecting SOD protein levels. We further demonstrate that O2·⫺ promotes, while H2O2 inhibits, NFATc3 activation in PASMC. Additionally, increased O2·⫺-to-H2O2 ratio activates NFATc3, even in the absence of a Gq protein-coupled receptor agonist. Furthermore, O2·⫺-dependent actin polymerization and low intracellular H2O2 concentration-dependent increases in intracellular Ca2⫹ concentration contribute to NFATc3 activation. Together, these studies define important and novel regulatory mechanisms of NFATc3 activation in PASMC by reactive oxygen species. superoxide; hydrogen peroxide; NFATc3; endothelin-1; actin cytoskeleton; calcium

T cells (NFAT) isoform c3 (NFATc3) belongs to a family of four transcription factors (NFATc1, NFATc2, NFATc3, and NFATc4) that share the property of Ca2⫹/calcineurin-dependent nuclear translocation (reviewed in Ref. 35). We previously demonstrated that NFATc3 is required for chronic hypoxia (CH)-induced pulmonary arterial remodeling and pulmonary hypertension in mice (10, 25). Pulmonary hypertension is associated with polycythemia, arterial remodeling, increased vascular contractility, augmented concentrations of circulating endothelin-1 (ET-1),

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Address for reprint requests and other correspondence: L. V. Gonzalez Bosc, Dept. of Cell Biology and Physiology, Univ. of New Mexico Health Sciences Center, MSC08 4750, Albuquerque, NM 87131 (e-mail: lgonzalezbosc@salud. unm.edu). C928

and elevated reactive oxygen species (ROS) (1, 9, 19, 24, 28, 32, 36, 55). ROS include, but are not limited to, superoxide (O2·⫺) and H2O2. Superoxide dismutase (SOD) is an antioxidant enzyme that catalyzes the dismutation of O2·⫺ into H2O2. Three SOD isoforms are expressed in the vasculature (2): SOD1 and extracellular SOD3 (Cu,Zn-SOD) and mitochondrial SOD2 (Mn-SOD); SOD1, the predominant cytosolic isoform (4), is also present in the mitochondrial intermembrane space (42). We recently showed that SOD1 knockout (KO) mice develop NFAT-dependent spontaneous pulmonary hypertension (62). CH-exposed wild-type and normoxic SOD1 KO mice have increased O2·⫺ and decreased H2O2 levels in pulmonary arteries. This O2·⫺-H2O2 imbalance is associated with elevated NFATc3 activity in pulmonary arterial smooth muscle cells (PASMC) (62). Similarly, in rats exposed to CH, we recently showed decreased pulmonary arterial SOD1 activity/expression, leading to increased O2·⫺ and decreased H2O2 production (59), as previously reported in newborn piglets (26). These findings suggest that an imbalance in ROS levels is required for NFATc3 activation. However, how such imbalance activates NFATc3 is unknown. We previously demonstrated that ET-1 contributes to CH-induced NFATc3 activation in PASMC (24). NFATc3 activation by ET-1 involves ET type A receptor (ETAR)mediated elevation of PASMC intracellular Ca2⫹ concentration ([Ca2⫹]i) and stimulation of RhoA/Rho kinase (ROCK) activity (24). [Ca2⫹]i activates calcineurin, which dephosphorylates NFATc3, exposing nuclear localization signals (reviewed in Refs. 35 and 63), and ROCK increases actin polymerization, providing the structural support for NFATc3 nuclear transport (24). The components of this signaling pathway are differentially regulated by both O2·⫺ and H2O2 (4, 5, 14, 40, 53). However, the role of O2·⫺ and/or H2O2 in the mechanisms of NFATc3 activation in PASMC has not been explored. Therefore, this study evaluated the importance of O2·⫺ and H2O2 in the regulation of NFATc3 activity, testing the hypothesis that an increase in O2·⫺ enhances actin cytoskeleton dynamics and a decrease in H2O2 enhances [Ca2⫹]i contributing to NFATc3 nuclear import and activation in PASMC. EXPERIMENTAL PROCEDURES

All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the University of New Mexico School of Medicine. Cell culture. Human PASMC (Life Technologies) were grown in poly-L-lysine (10 ␮g/ml)-coated flasks in Growth Medium 231 (Life

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O2·⫺ AND H2O2 HAVE OPPOSITE EFFECTS ON NFATc3

Technologies) at 37°C in 5% CO2 with controlled humidity. Before any experiment, cells were cultured for ⱖ48 h in a differentiation medium that contained 1% FBS and 30 ␮g/ml heparin (Life Technologies). In all experiments, cells were preincubated with a myosin light chain kinase peptide inhibitor (1 ␮M) to prevent cell contraction and detachment. O2·⫺ detection by spin trapping combined with electron paramagnetic resonance spectroscopy. Generation of O2·⫺ by human PASMC was monitored using electron paramagnetic resonance (EPR) spectroscopy combined with spin trapping, as reported in the literature with a slight modification (66, 68). The nitrone 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide (BMPO; Enzo Life Sciences) was used as the spin trap for O2·⫺ generated from cells. In a typical experiment, vehicle (water) or ET-1 (100 nM, final concentration) was added to a reaction system that contained BMPO (50 mM, final concentration), diethylenetriaminepentaacetic acid (100 ␮M final concentration), and PASMC (⬃106 cells). This reaction system was incubated for 30 min at 37°C. After the incubation period, medium (400 ␮l) containing spin-trapped O2·⫺ was immediately transferred to custom-made gaspermeable Teflon tubing (Zeus Industries, Raritan, NJ), folded four times, and inserted into a quartz EPR tube open at each end. The quartz EPR tube was inserted into the cavity of an EPR spectrometer (EleXsys 540 X-band, Bruker, Billerica, MA) operating at 9.8 GHz and 100-kHz field modulation, and the spectra of BMPO-OOH, spin-trapped O2·⫺, was recorded after spectrometer tuning at room temperature. The EPR spectrum was acquired with a scan time of 40 s, and 10 scans were obtained and averaged to produce significant signal-to-noise ratio. Instrument settings were as follows: magnetic field, 3,509 G; scan range, 120 G; microwave power, 21 mW; modulation frequency, 100 kHz; modulation amplitude, 1.0 G; time constant, 20 ms. The EPR spectra were collected, stored, and manipulated using Xepr software (Bruker). Polyethylene glycol (PEG)-SOD (120 U/ml final concentration) was used to confirm that O2·⫺ was the free radical detected by EPR. H2O2 detection with the H2O2 sensor HyPer. Human PASMC were electroporated with the vector HyPer (Evrogen), which encodes a fluorescent sensor capable of detecting intracellular H2O2. HyPer demonstrates submicromolar affinity to H2O2; at the same time, it is insensitive to other oxidants tested, such as O2·⫺, oxidized glutathione, nitric oxide, and peroxinitrite (11, 51). Fluorescence intensity (488-nm excitation, 516-nm emission) was monitored every 30 s using a Nikon Diaphot 300 microscope at ⫻200 magnification. Fluorescence was background-corrected and expressed as fold change from baseline fluorescence (F0), calculated from the average of at least six frames (F/F0; Metamorph Universal Imaging software). H2O2 detection by Amplex Red/peroxidase. H2O2 production was determined by the Amplex Red/peroxidase assay (Life Technologies) in cultured human PASMC and intrapulmonary (2nd- and 3rd-order, ⬃4-mm-long) arteries (62) incubated in HEPES-physiological saline solution (PSS) for 30 min. The H2O2 concentration was calculated from a standard curve and normalized by total protein measured by Bradford assay (Bio-Rad) or DNA (fluorescence intensity of SYTOX staining). Briefly, human PASMC were seeded on a poly-L-lysinecoated black 96-well clear flat-bottom plate (BD Falcon), grown, and differentiated as described in Cell culture. On the day of the experiment, cells or arteries were treated as described in RESULTS, and fluorescence intensity (550-nm excitation, 610-nm emission) was measured with a Tecan 200 plate reader 30 min after the addition of Amplex Red (5 mM final concentration) and horseradish peroxidase (5 U/ml final concentration). Background was determined in a well without cells/arteries but with HEPES-PSS buffer and Amplex Red/ horseradish peroxidase. PEG-catalase (200 U/ml) was used to determine the specificity of the reaction, as previously described (62). In-gel SOD activity assay. The SOD activity gel assay is based on inhibition of nitro blue tetrazolium (NBT) reduction by SOD. The principle of this assay is based on the ability of O2·⫺ to interact with NBT, reducing the yellow tetrazolium within the gel to a blue

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precipitate. Areas where SOD is active develop a clear area (achromatic bands) competing with NBT for the O2·⫺ (7). Briefly, human PASMC were cultured in poly-L-lysine-coated 175-cm2 flasks, as described in Cell culture. After treatment (see RESULTS), cells were washed with cold HEPES-PSS and lysed with an isotonic Ca2⫹-free HEPES buffer (in mM: 20 HEPES, 1 EGTA, 210 mannitol, and 70 sucrose). Proteins were then separated by native PAGE (Bio-Rad), and gels were stained with a solution of 2.3 mM NBT in distilled water, incubated for 15 min with a solution containing 0.028 mM riboflavin and 280 mM tetramethylethylenediamine in PBS, and exposed to light for 7 min. PEG-SOD (60 U/ml) was used as the positive control. Half of the samples and gels were incubated with the SOD1 inhibitor diethyldithiocarbamate (DDC, 100 ␮M) (38). Protein loading was determined by staining the gels with Coomassie blue (Bio-Rad). Gels were imaged with a HP Officejet Pro 8500A scanner and analyzed with ImageJ (National Institutes of Health). SOD isoform levels by Western blotting. An aliquot of the samples used to determine SOD activity (40 ␮g/lane) was resolved by SDSPAGE, and proteins were transferred to PVDF membranes. The membranes were blocked with blocking buffer (Odyssey, LI-COR Biosciences) and then incubated with primary antibody [rabbit antiSOD1 (catalog no. ab13498, Abcam), rabbit anti-SOD2 (catalog no. ab13534, Abcam), or rabbit anti-SOD3 (catalog no. ab83103, Abcam) and mouse anti-␤-actin (Sigma)] at 1:5,000 dilution in 0.1% Tween PBS at 4°C overnight, washed, and incubated for 1 h with 1:10,000diluted goat anti-rabbit IRdye800cw and goat anti-mouse IRdye680 (LI-COR Biosciences). The membrane was scanned using the Odyssey infrared imaging system (LI-COR Biosciences). Results are expressed as the ratio of SOD to ␤-actin fluorescence intensity. NFATc3 nuclear translocation. PASMC were electroporated (Nucleofector, Lonza) with NFATc3-enhanced green fluorescent protein (EGFP) expression vector, which was created by Dr. F. McKeon (Harvard University, Cambridge, MA) and kindly provided by Dr. L. F. Santana (Washington State University, Seattle, WA). Electroporated cells were seeded on microscope coverslips coated with poly-L-lysine and cultured in growth medium for 24 h and then in differentiation medium for ⱖ48 h. Then cells were subjected to different treatments (see RESULTS), fixed with 4% formaldehyde in PBS at the end of the treatment, washed with PBS and water, and imaged using a ⫻63 objective on a Leica TCS SP5 spectral confocal system. Nuclear EGFP and cytosol EGFP fluorescence (FN and FC, respectively) were measured by placing regions of interest in the nucleus and cytosol with Leica LAS AF Lite software. Fluorescence values were background-corrected and are expressed as FN/FC. Animals. Adult male 9x-NFAT-luciferase reporter (NFAT-luc) mice (20 –25 g) were provided by Dr. Jeffery D. Molkentin (Department of Pediatrics, Children’s Hospital Medical Center, Cincinnati, OH) (13). All animals were maintained on a 12:12-h light-dark cycle. Luciferase activity. NFAT-luc mice were anesthetized with an overdose of pentobarbital solution (200 mg/kg ip). Lungs were removed and placed into HEPES-PSS (in mM: 134 NaCl, 6 KCl, 1 MgCl, 10 HEPES, 2 CaCl2, 0.026 EDTA, and 10 glucose). Intrapulmonary arteries were isolated, cultured for 24 h (see RESULTS), and lysed using tissue lysis buffer (Promega) according to the manufacturer’s protocol. The lysate was centrifuged for 10 min at 10,000 relative centrifugal force. Luciferase activity and protein content were determined in the supernatant. Luciferase activity was measured using a Luciferase Assay System kit (Promega), and light was detected with a luminometer (model TD20/20, Turner). Protein content was determined by the Bradford method (Bio-Rad) and used to normalize luciferase activity per sample. [Ca2⫹]i measurement. Human PASMC were seeded on poly-Llysine-coated coverslips and grown as described in Cell culture. Cells were incubated at room temperature with HEPES-PSS containing the cell-permeable ratiometric Ca2⫹-sensitive fluorescent dye fura 2-acetoxymethyl ester (fura 2-AM, 2 ␮M; Life Technologies) and 0.02% pluronic acid (Life Technologies) for 15 min. Then cells were washed

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with warm HEPES-PSS (37°C) multiple times and equilibrated for an additional 15 min. Fura 2-AM-loaded cells were alternately excited at 340 and 380 nm at a frequency of 10 Hz with an IonOptix Hyperswitch dual-excitation light source, and the respective 510-nm emissions were collected with a photomultiplier tube [ratio of fluorescence at 340 nm to fluorescence at 380 nm (F340/F380)]. After subtraction of background fluorescence (which was collected before fura loading), emission ratios were calculated with IonWizard software (IonOptix) and recorded continuously throughout the experiment. Percent changes in F340/F380 in response to treatments were calculated from baseline. Actin cytoskeleton polymerization. Human PASMC were treated as described in RESULTS, formaldehyde-fixed (4% in PBS), permeabilized with 0.1% Triton X-100 in PBS, and stained for filamentous (F)-actin with Alexa Fluor 488-phalloidin (6.6 ⫻ 10⫺6 M; Life Technologies) and for globular (G)-actin with Alexa Fluor 594-DNase I (0.64 ⫻ 10⫺6 M; Life Technologies) at room temperature. Cells were imaged with a ⫻63 objective on a Leica TCS SP5 spectral confocal system. F-actin and G-actin intensity (arbitrary units) was measured by tracing lines across multiple cells per field using Leica LAS AF Lite software and expressed as a ratio. NFATc2 and NFATc3 mRNA levels. Isolated intrapulmonary arteries were stored in RNAlater (Ambion). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) and reverse-transcribed to cDNA using a High Capacity Reverse Transcription kit (Life Technologies). NFATc2 and NFATc3 transcript levels were measured using premade TaqMan Assays (Life Technologies). The normalized gene expression [cycle threshold (CT)] method (2⫺⌬⌬CT) for relative quantification of gene expression was used (50). ␤-Actin was used as the endogenous control. Sample size and statistical analysis. When cultured cells were used, experiments were repeated at least three times with different batches of cells. In experiments with isolated intrapulmonary arteries, arteries from one mouse were subjected to a set of treatments, including a control, and then at least three animals were used. Values are means ⫾ SE. Statistical significance was tested at 95% (P ⬍ 0.05) confidence level using unpaired t-test, one-way ANOVA, Kruskal-Wallis ANOVA on ranks, or two-way ANOVA followed by Newman-Keuls multiple comparisons test. RESULTS

ET-1 increases O2·⫺ and decreases H2O2 by decreasing SOD1 activity. Previous reports demonstrate that ET-1 increases O2·⫺ production (40, 54, 72, 73). Similarly, we found that 30 min of incubation with ET-1 (100 nM) increased O2·⫺ production in human PASMC, measured by the increase in BMPO-OOH EPR signal in the incubation medium (Fig. 1A). Preincubation with PEG-SOD (120 U/ml) for 30 min and during the 30-min control and ET-1 treatment completely eliminated the BMPO-OOH EPR signal. Additionally, PEGcatalase did not affect the basal BMPO-OOH EPR signal in human PASMC (data not shown), confirming the specificity of the assay. These results confirm that the detected free radical was O2·⫺ and demonstrate that ET-1 increases O2·⫺ production in human PASMC. In cells, O2·⫺ is rapidly dismutated to H2O2 by SOD. Since controversy exists on the effect of ET-1 on cellular H2O2 production (41, 58, 61, 74), we measured H2O2 by two independent methods. In human PASMC expressing the H2O2 sensor HyPer, we found that ET-1 significantly decreased intracellular H2O2 levels, reaching a plateau in ⬃10 min (Fig. 1B). Using the H2O2-sensitive probe Amplex Red, we also determined the effect of ET-1 on extracellular H2O2 production. We found a significant decrease in H2O2 production in

human PASMC and mouse pulmonary arteries upon 30 min of ET-1 (100 nM) stimulation (Fig. 1C). Additionally, in human PASMC, the SOD1 inhibitor DDC (10 ␮M) significantly decreased extracellular H2O2 concentration in control cells. Interestingly, DDC did not further decrease H2O2 in ET-1treated cells and normalized H2O2 levels between groups. On the contrary, a catalase inhibitor, aminotriazole (5 ␮M), increased extracellular H2O2 concentration in control cells but did not affect ET-1-induced decreases in H2O2, suggesting that ET-1 does not affect catalase activity (Fig. 1C). Addition of exogenous PEG-catalase (200 U/ml) significantly decreased extracellular H2O2 concentration in control cells and showed a trend in ET-1-treated cells confirming the specificity of the assay (Fig. 1C). These results suggest that the decrease in H2O2 production induced by ET-1 is mediated by the inhibition of SOD1 activity and not an increase in catalase activity (Fig. 1C). To assess the mechanism of the ET-1-induced increase in O2·⫺ and decrease in H2O2, we used an in-gel activity assay to determine SOD activity in human PASMC. Cells incubated with ET-1 for 5 min show a significant decrease in total SOD activity compared with control cells (Fig. 2). Additionally, the SOD1 inhibitor DDC (1 mM) reduced activity in control cells without further decreasing SOD activity in ET-1-treated cells, thereby normalizing activity within groups (Fig. 2B). These data suggest that ET-1 inhibits SOD1 activity. To rule out a possible effect of ET-1 on SOD enzyme levels, SOD1–3 protein levels were determined by Western blotting in the same samples used to measure enzyme activity (Fig. 3). As expected, no significant differences were observed in the levels of the different SOD isoforms between ET-1- and vehicletreated human PASMC (Fig. 3). ET-1-induced NFATc3 nuclear import and transcriptional activity depend on a balance of O2·⫺ and H2O2. We assessed the effect of ET-1-induced increases in the O2·⫺-to-H2O2 ratio on NFATc3 nuclear import and activity on human PASMC and mouse pulmonary arteries, respectively. To determine NFATc3 nuclear import, nuclear export was blocked with the chromosomal region maintenance-1 (CRM1) exportin inhibitor leptomycin B (40 nM). Before ET-1 stimulation, the cells were preincubated with tempol (3 mM) for 30 min or PEG-SOD (120 U/ml) for 1 h to decrease O2·⫺, with PEG-catalase (250 U/ml) for 30 min to decrease H2O2, and with exogenous H2O2 (200 ␮M). Arteries were incubated ex vivo for 24 h with the same compounds and concentrations, but in Opti-MEM (Life Technologies). We found that ET-1-induced NFATc3 nuclear import and transcriptional activity were prevented by addition of exogenous H2O2 (Figs. 4A and 5A) or by decrease in O2·⫺ with tempol or PEG-SOD (Fig. 4, A and B, and Fig. 5A). On the contrary, decreasing H2O2 with PEG-catalase enhanced ET-1-induced NFATc3 nuclear import in PASMC (Fig. 4A) and transcriptional activity in pulmonary arteries (Fig. 5A). Furthermore, decreasing O2·⫺ with tempol prevented the enhanced ET-1-induced NFAT activation caused by PEG-catalase (Figs. 4A and 5A). These results suggest that O2·⫺ and H2O2 have opposite effects on NFATc3 activation and that NFAT activation requires basal O2·⫺ levels when H2O2 is decreased. None of the pretreatments, including PEG-catalase, affected basal NFATc3 nuclear levels in PASMC (data not shown). However, PEG-catalase increased NFAT transcriptional activ-

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Fig. 1. Endothelin 1 (ET-1) increases O2·⫺ and decreases H2O2 by inhibiting SOD1 in pulmonary arterial smooth muscle cells (PASMC). A: O2·⫺ detected by spin-trapping electron paramagnetic resonance in cultured human PASMC treated for 30 min with or without ET-1 (100 nM) and pretreated with vehicle (water) or polyethylene glycol (PEG)-SOD (120 U/ml) for 1 h in a HEPES-based buffer. Signal-to-noise ratio (SNR) was normalized by the total number of cells and expressed as fold change from control (vehicle-treated cells). Values are means ⫾ SE; n ⫽ 4 times. *P ⬍ 0.001 vs. control; #P ⬍ 0.0001 vs. vehicle (by Kruskal-Wallis ANOVA on ranks followed by Student-Newman-Keuls method). B: intracellular H2O2 detected in cultured human PASMC electroporated with the H2O2 sensor HyPer. Top: pseudocolored images of representative cells at baseline and after treatment with ET-1 (100 nM) or H2O2 (200 ␮M). Bottom: representative traces and summary data of fluorescence (F) intensity in regions of interest background-corrected and divided by the average of ⱖ6 baseline frames (F0). Values are means ⫾ SE; n ⫽ 4 –10 times, 2–3 cells per field. *P ⬍ 0.05 (by Student’s t-test). C: extracellular H2O2 detected with the Amplex Red/peroxidase assay in cultured human PASMC and isolated intrapulmonary arteries. Cells were pretreated with vehicle, diethyldithiocarbamate (DDC, 10 ␮M), aminotriazole (AT, 5 ␮M), or PEG-catalase (200 U/ml) for 1 h and then incubated with water (control) or ET-1 (100 nM) for 30 min. Cells were also stained with SYTOX green to account for differences in the number of cells per well. H2O2 concentration was then normalized to the SYTOX signal or to total artery protein. Values are means ⫾ SE; n ⫽ 4 –10 times with ⬃10,000 cells per well; n ⫽ 4 arteries from 4 animals. *P ⬍ 0.05 vs. control, #P ⬍ 0.05 vs. vehicle (by 2-way ANOVA followed by Student-Newman-Keuls method).

ity in pulmonary arteries (Fig. 5A). To determine why decreasing H2O2 with PEG-catalase activated NFAT in pulmonary arteries but did not cause NFATc3 nuclear import in PASMC, leptomycin B was added simultaneously with PEG-catalase,

and the PASMC were incubated for 1 h, instead of 30 min, resulting in NFATc3 nuclear import (Fig. 4C). To determine whether altering the balance of O2·⫺ and H2O2 is enough to activate NFAT, we cultured pulmonary arteries

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Fig. 2. ET-1 decreases SOD activity in PASMC. A: representative images of in-gel determination of SOD activity in cultured human PASMC treated with (ET) or without [control (C)] ET-1 for 5 min. Samples were resolved by nondenaturing electrophoresis in the absence (vehicle) or presence of DDC (100 ␮M). Equal protein loading was verified by staining the gel with Coomassie blue. B: summary of data normalized to control. Values are means ⫾ SE; n ⫽ 5. *P ⬍ 0.05 vs. vehicle control, #P ⬍ 0.05 vs. vehicle ET (by Kruskal-Wallis ANOVA on ranks followed by Student-Newman-Keuls method).

with the O2·⫺ generator SOTS-1 (100 ␮M) in the presence or absence of PEG-catalase. Increased O2·⫺ alone had no significant effect, but enhanced PEG-catalase induced NFAT activation in pulmonary arteries (Fig. 5B). These findings suggest that an increase in O2·⫺, together with a decrease in H2O2 levels, is sufficient to activate NFAT, further confirming that O2·⫺ and H2O2 have opposite effects on NFAT activation.

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NFATc3 nuclear export and JNK1/2 are not involved in the mechanism of NFATc3 activation induced by elevated O2·⫺to-H2O2 ratio. We determined whether the ET-1-induced elevated O2·⫺-to-H2O2 ratio affects NFATc3 nuclear export. Translocation of the transcription factor was induced by 5 min of incubation with ET-1 (100 nM), and subsequent import was inhibited by addition of the calcineurin inhibitor cyclosporin A (CsA, 1 ␮M). The experiment was performed in the presence of tempol (3 mM). Figure 6A shows that decreasing O2·⫺ with tempol does not affect NFATc3 nuclear export. We previously demonstrated that c-Jun-NH2-terminal kinase (JNK2) phosphorylates NFATc3, enhancing its nuclear export (3, 33). As a positive control, we determined NFATc3 nuclear export in the presence of a JNK1/2 inhibitor, SP600125 (0.08 ␮M; Calbiochem). As expected, the JNK1/2 inhibitor attenuated NFATc3 nuclear export (Fig. 6A). These experiments were not performed in the presence of PEG-SOD or PEG-catalase, because these compounds require preincubation to allow for their cellular uptake. These results suggest that alterations in O2·⫺H2O2 balance do not affect the mechanisms that regulate NFATc3 nuclear export. JNK2 is also in the cytosol, and ET-1-induced increases in the O2·⫺-to-H2O2 ratio could be inhibiting JNK2 activity as part of the mechanism of NFATc3 activation. To address this possibility, the effect of tempol and exogenous H2O2 on ET-1-induced NFATc3 nuclear import was determined in the absence or presence of the JNK1/2 inhibitor. Figure 6B shows that, as expected, the JNK1/2 inhibitor enhanced ET-induced NFATc3 nuclear import but did not affect the inhibitory effect of decreasing O2·⫺ with tempol or exogenous H2O2. In summary, these data suggest that JNK2 is not involved in the mechanism of NFATc3 activation induced by an elevated O2·⫺-to-H2O2 ratio. A decrease in H2O2 increases [Ca2⫹]i and activates NFATc3 in a Ca2⫹/calcineurin-dependent manner. Our results demonstrate that reduced levels of H2O2, together with basal levels of O2·⫺, are sufficient to induce NFATc3 nuclear import (Figs. 4C and 5). Since calcineurin-mediated NFAT dephosphorylation is a required step in the NFAT activation pathway, we determined whether degradation of H2O2 with PEG-catalase increases [Ca2⫹]i. Figure 7A shows fura 2 F340/F380 emission ratios from cells treated with PEG-catalase (250 U/ml) before (45 min) and during (15 min) fura 2 loading and

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Fig. 3. ET-1 does not affect SOD isoform protein levels. A: representative immunoblot detection of SOD1, SOD2, SOD3, and ␤-actin levels in cultured human PASMC treated with or without [control (C)] ET-1. B: summary of the analysis of the density of the bands normalized to ␤-actin. AJP-Cell Physiol • doi:10.1152/ajpcell.00244.2014 • www.ajpcell.org

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O2·⫺ AND H2O2 HAVE OPPOSITE EFFECTS ON NFATc3

NFATc3-EGFP FN/FC

10 8

*

6 4

*

# #

#

2

Control ET Tempol + ET H2O2 + ET Cat + ET Cat + tempol + ET

#

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8 6

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Ca2⫹-ATPase (SERCA) inhibitor cyclopiazonic acid and preventing extracellular Ca2⫹ influx abolished the activation of NFAT induced by decreasing H2O2 with PEG-catalase (Fig. 7D). These findings suggest that Ca2⫹-dependent calcineurin is required for NFAT activation during low levels of H2O2. Since we recently demonstrated that PEG-catalase activates acid-sensing ion channel 1 (ASIC1) in PASMC (59) and these channels conduct Ca2⫹ (39), we tested whether Ca2⫹ influx through ASIC1 contributes to PEG-catalase-induced NFATc3 nuclear import. We found that ASIC1 inhibition with psalmotoxin 1 (100 ng/ml) prevented PEG-catalase-induced NFATc3 nuclear import (Fig. 7E), suggesting that ASIC1 activation by low levels of H2O2 contributes to NFATc3 nuclear import. O2·⫺ is not required for ET-1-induced increases in [Ca2⫹]i. Our data indicate that O2·⫺ is required for ET-1-induced NFATc3 nuclear import (Fig. 4, A and B) and transcriptional activity (Fig. 5A) but is not sufficient to activate NFAT (Fig. 5B). Therefore, we tested whether O2·⫺ is also required for ET-1-induced increases in [Ca2⫹]i. Figure 8 shows that pretreatment of human PASMC with tempol (3 mM) does not affect the peak increase in fura 2 F340/F380 emission ratios or the plateau in response to ET-1. These results suggest that O2·⫺ does not affect the ET-1-induced Ca2⫹ response. O2·⫺ is required for ET-induced increases in cytoskeletal actin polymerization. We previously demonstrated that a dynamic actin cytoskeleton is required for ET-1-induced NFATc3 nuclear import (24). The cytoskeleton of smooth

2

A

0

Fig. 4. ET-1-induced nuclear factor of activated T cells (NFAT) isoform c3 (NFATc3) nuclear import depends on O2·⫺-H2O2 balance. A: enhanced green fluorescent protein (EGFP)-NFATc3 nuclear and cytosolic fluorescence (FN and FC, respectively) was determined in cultured human PASMC pretreated with vehicle, tempol (3 mM), H2O2 (200 ␮M), or PEG-catalase (200 U/ml) for 30 min followed by no additional treatment or treatment with ET-1 (100 nM) for 30 min. B: cells were treated with PEG-SOD (120 U/ml) for 1 h before addition of ET-1. C: cells were treated with PEG-catalase (cat) for 1 h before addition of ET-1. Values are means ⫾ SE; n ⫽ 4 –9. *P ⬍ 0.05 vs. control, #P ⬍ 0.05 vs. ET-1 (by ANOVA followed by Student-Newman-Keuls method).

Luciferase Activity RLU.μg-1

A

80

*

Control ET ET + H2O2 ET + tempol Cat Cat + ET Cat + tempol + ET Cat + tempol

#&

60

40

20

* *

* #

&

#

0

B Luciferase Activity RLU.μg-1

in vehicle-treated cells. F340/F380 emission ratios were significantly higher than in cells treated with vehicle (water). Additionally, Fig. 7B shows a significant increase in F340/F380 emission ratios from baseline in cells first loaded with fura 2 and then treated with PEG-catalase for 1 h. Since our results suggest that a decrease in H2O2 increases [Ca2⫹]i in PASMC, we assessed the contribution of this increase in [Ca2⫹]i to NFAT activation. We determined NFATc3 nuclear import in human PASMC treated with PEG-catalase in the absence or presence of low extracellular Ca2⫹ ([Ca2⫹]e ⫽ 100 nM), diltiazem (50 ␮M), or CsA (1 ␮M). Figure 7C shows that low [Ca2⫹]e or calcineurin inhibition (CsA) prevented PEG-catalase-induced NFATc3 nuclear import. Inhibition of L-type voltage-gated Ca2⫹ channels also diminished NFATc3 nuclear import; however, diltiazem was significantly less effective than low [Ca2⫹]e. Similarly, pulmonary arteries were incubated ex vivo for 24 h with PEG-catalase in the absence or presence of low [Ca2⫹]e (100 nM) plus cyclopiazonic acid (10 ␮M) and diltiazem (50 ␮M). We found that depleting intracellular Ca2⫹ stores with the sarco/endoplasmic reticulum

*#

30

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*

Control Catalase SOTS-1 SOTS-1+catalase

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Fig. 5. An increase in O2·⫺ together with a decrease in H2O2 levels is sufficient to activate NFAT. A: intrapulmonary arteries were isolated from NFATluciferase (NFAT-luc) mice and incubated for 30 min with vehicle, tempol (3 mM), H2O2 (200 ␮M), or PEG-catalase (200 U/ml) followed by no additional treatment or treatment with ET-1 for 24 h. RLU, relative light units. Values are means ⫾ SE; n ⫽ 3– 6. *P ⬍ 0.05 vs. control, #P ⬍ 0.05 vs. ET-1, &P ⬍ 0.05 vs. catalase (by ANOVA followed by Student-Newman-Keuls method). B: arteries were incubated with SOTS-1 (100 ␮M) in the presence or absence of PEG-catalase. Values are means ⫾ SE; n ⫽ 3– 6 animals. *P ⬍ 0.05 vs. control, #P ⬍ 0.05 vs. catalase (by 2-way ANOVA followed by StudentNewman-Keuls method).

AJP-Cell Physiol • doi:10.1152/ajpcell.00244.2014 • www.ajpcell.org

C934

O2·⫺ AND H2O2 HAVE OPPOSITE EFFECTS ON NFATc3

NFATc3-EGFP FN/FC

A

2.0

Control Tempol JNKi

*

1.5 1.0 0.5 0.0

1

5

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Time (minutes)

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50

DISCUSSION

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Control ET H2O2 + ET Tempol + ET

40 30

*

20 10 0

*#

*# # Vehicle

for 24 h with vehicle (water), ET-1 (100 nM), PEG-catalase (250 U/ml), or ET-1 and PEG-catalase. Neither NFATc2 (Fig. 10A) nor NFATc3 (Fig. 10B) mRNA levels were affected by these treatments, suggesting that a change in NFAT isoform expression is not the underlying mechanism of NFAT activation (Fig. 5A) under the same experimental conditions. NFAT isoform mRNA levels were not measured in cultured PASMC, because none of the treatments exceeded 1 h; therefore, it is unlikely that de novo synthesis could have occurred in such a short period of time.

#

#

JNKi

Fig. 6. NFATc3 nuclear export and JNK1/2 are not involved in the mechanism of NFATc3 activation induced by an elevated O2·⫺-to-H2O2 ratio. A: EGFPNFATc3 FN and FC were determined over time in cultured human PASMC in which nuclear import was induced with ET-1 (100 nM) and export was initiated by addition of cyclosporin A (CsA, 1 ␮M) with or without tempol (3 mM) or the JNK1/2 inhibitor SP600125 (0.08 ␮M). Values are means ⫾ SE; n ⫽ 3. *P ⬍ 0.05 vs. control (by Kruskal-Wallis ANOVA on ranks followed by Student-Newman-Keuls method). B: EGFP-NFATc3 FN and FC were determined in cultured human PASMC pretreated with or without H2O2 (200 ␮M) or tempol (3 mM) in the presence or absence of SP600125 (0.08 ␮M) followed by no additional treatment or ET-1 (100 nM). Values are means ⫾ SE; n ⫽ 4. *P ⬍ 0.05 vs. control, #P ⬍ 0.05 vs. ET-1, &P ⬍ 0.05 vs. ET-1 vehicle (by 2-way ANOVA followed by Student-Newman-Keuls method).

muscle cells is a dynamic filamentous network consisting largely of F-actin. Therefore, we tested whether an increase in O2·⫺ and/or a reduction in H2O2 affects cytoskeletal actin polymerization by assessing the ratio of F- to G-actin. Figure 9 shows that, as previously demonstrated (24), ET-1 (100 nM, 5 min) increased the ratio of F- to G-actin compared with control. Interestingly, we found that this increase is prevented by decreasing O2·⫺ with tempol (3 mM) or PEG-SOD (120 U/ml) but is not affected by reducing H2O2 with PEG-catalase (250 U/ml). Neither tempol nor PEG-SOD or PEG-catalase affected the basal ratio of F- to G-actin (data not shown). Neither ET-1 nor PEG-catalase affects NFATc2 and NFATc3 expression in pulmonary arteries. We have shown that NFATc3, but not NFATc2, is activated in PASMC by hypobaric CH and in SOD1 KO mice (24, 25, 62). However, NFATc2 has been shown to be activated in PASMC from patients with primary pulmonary arterial hypertension (PAH) and in other animal models of PAH (12, 21, 22, 52, 57, 67). NFATc2 activation is regulated at the level of its expression through the signal transducers and activators of transcription-3 (STAT3) and by the oncoprotein kinase Pim1 pathway (12, 21, 22, 52, 57, 67). To determine whether ET-1 or PEG-catalase treatment affects NFATc2 or NFATc3 expression, NFATc2 and NFATc3 mRNA levels were determined by real-time PCR in isolated pulmonary arteries. Arteries were incubated ex vivo

This study demonstrates that, in PASMC, ET-1 increases O2·⫺ while decreasing H2O2 production through the decrease in SOD1 activity without affecting SOD protein levels. This study further demonstrates that O2·⫺ promotes, while H2O2 inhibits, NFATc3 activation in PASMC. Additionally, we demonstrate that increased O2·⫺-to-H2O2 ratio activates NFATc3, even in the absence of a Gq protein-coupled receptor agonist. Furthermore, it demonstrates that O2·⫺-dependent actin polymerization and low intracellular H2O2-dependent increases in [Ca2⫹]i contribute to NFATc3 activation. It is well established that ETAR activation by ET-1 increases O2·⫺ production by enhancing NADPH oxidase activity in PASMC (40, 54, 72, 73). However, the effect on H2O2 production is less clear. It has been reported that ET-1 elevates H2O2 in lung and PASMC (58, 74). However, it has also been reported that ET-1 decreases H2O2 in melanocytes (41) and pulmonary endothelial cells (61). Our results showing that ET-1 enhances O2·⫺ production but decreases H2O2 are consistent with some of these previous reports. However, this is the first report showing that ET-1 reciprocally changes O2·⫺ and H2O2 levels in PASMC. One possibility for the reciprocal levels of O2·⫺ and H2O2 following ET-1 treatment is the inhibition of SOD activity. We have shown the same alteration in the O2·⫺-to-H2O2 ratio in pulmonary arteries of SOD1 KO mice (62) and rats exposed to CH (59). Furthermore, it has been shown that ET-1 reduces SOD activity and expression in the systemic vasculature (15, 16). Consistent with this possibility, our data demonstrate that ET-1 reduces SOD activity in PASMC. However, ET-1 did not affect SOD protein levels. The lack of change in protein levels could be due to the short-duration (5-min) ET-1 treatment. DDC, a selective inhibitor of SOD1, reduced SOD activity in control cells and did not further affect ET-1-induced decreases in SOD activity, suggesting that the isoform regulated by ET-1 is SOD1. An additional mechanism that could contribute to ET-1-induced reduction of H2O2 levels would be the increased activity of the enzymes responsible for H2O2 degradation: catalase and glutathione peroxidase (6). ET-1 has been shown to decrease H2O2 by increasing catalase activity in pulmonary endothelial cells (46, 61). However, our data suggest that ET-1 does not affect catalase activity, because ET-1 still reduced H2O2 production while catalase was inhibited. In addition, SOD1 inhibition in control cells mimicked the effect of ET-1 and normalized H2O2 levels between groups, suggesting that decreased SOD1 activity is the main mechanism. However, we cannot discard the possibility that ET-1 could have enhanced glutathione peroxidase activity. In addition, it is possible that animal models or diseases in which SOD2 activity is decreased

AJP-Cell Physiol • doi:10.1152/ajpcell.00244.2014 • www.ajpcell.org

C935

O2·⫺ AND H2O2 HAVE OPPOSITE EFFECTS ON NFATc3

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Fig. 7. A decrease in H2O2 increases intracellular Ca2⫹ concentration ([Ca2⫹]i) and activates NFATc3 in a Ca2⫹/calcineurin-dependent manner. A: fura 2 ratio of fluorescence at 340 nm to fluorescence at 380 nm (F340/F380) from cells treated with PEG-catalase (200 U/ml) before (45 min) and during (15 min) fura 2 loading and compared with vehicle-treated cells. Values are means ⫾ SE; n ⫽ 4. *P ⬍ 0.05 (by Student’s t-test). B: fura 2 F340/F380 values from baseline in cells first loaded with fura 2 and then treated with PEG-catalase for 1 h. Values are means ⫾ SE; n ⫽ 3. *P ⬍ 0.05 (by Student’s t-test). C: EGFP-NFATc3 FN and FC in cultured human PASMC treated with PEG-catalase (200 U/ml) in the absence or presence of low extracellular Ca2⫹ (100 nM), diltiazem (50 ␮M), or CsA (1 ␮M). Values are means ⫾ SE; n ⫽ 4. *P ⬍ 0.05 vs. control, #P ⬍ 0.05 vs. vehicle. 2-way ANOVA followed by StudentNewman-Keuls method. D: intrapulmonary arteries were isolated from NFAT-luc mice and incubated for 24 h with PEGcatalase in the absence or presence of low extracellular Ca2⫹ (100 nM) plus cyclopiazonic acid (CPA, 10 ␮M) and diltiazem (Dilt, 50 ␮M). Values are means ⫾ SE; n ⫽ 4 animals. *P ⬍ 0.05 vs. control, #P ⬍ 0.05 vs. catalase (by ANOVA followed by Student-Newman-Keuls method). E: EGFP-NFATc3 FN/FC in cultured PASMC treated with PEG-catalase (200 U/ml) in the absence or presence of psalmotoxin 1 (PcTx1, 100 ng/ml). Values are means ⫾ SE; n ⫽ 4. *P ⬍ 0.05 vs. control, #P ⬍ 0.05 vs. catalase (by ANOVA followed by Student-NewmanKeuls method).

#

0

lation by several serine/threonine kinases, such as glycogen synthase kinase-3␤ and JNK1/2 (3, 33–35), and by the activity of the exportin CRM1 (3, 33, 35). The components of this signaling pathway, except for CRM1, have been shown to be differentially regulated by O2·⫺ and H2O2 (4, 5, 14, 17, 18, 40, 49, 53, 64, 71). Our study shows that NFATc3 nuclear export was not affected by changes in the O2·⫺-to-H2O2 ratio. In addition, our results demonstrate that JNK1/2 regulates NFATc3 nuclear export and import in PASMC, as previously described in other cell types (3, 33–35). Furthermore, the effects of O2·⫺ and

20

% F340/F380 baseline

could be associated with increased NFAT activity, since H2O2 levels are decreased and [Ca2⫹]i is increased in those conditions (4). Our data in PASMC suggest that O2·⫺ and H2O2 have opposite effects on NFATc3 activation by ET-1. This conclusion is based on our finding that O2·⫺ scavengers (tempol and PEG-SOD) prevented ET-1-induced NFATc3 activation, and a O2·⫺ generator enhanced and tempol inhibited PEG-catalase-induced NFATc3 activation. These results also suggest that low H2O2 levels with basal O2·⫺ production are sufficient to activate NFATc3. In addition, our study shows that H2O2 plays an inhibitory role, because exogenous H2O2 inhibits, while degradation of H2O2 enhances, ET-1-induced NFATc3 activation. This was similarly observed in mouse Cl41 and human lung bronchial epithelial A549 cells (43). In these cells, O2·⫺ is responsible for silica-induced NFAT activation. In addition, H2O2 degradation with catalase enhances silica-induced NFAT activation in these cells (43), consistent with H2O2 inhibition of NFAT (8, 29, 31, 56) but in contrast to reports showing that H2O2 activates NFAT in immune cells (30, 37, 44, 69, 70). Current dogma suggests that Ca2⫹ influx and/or release from the endoplasmic reticulum activates calcineurin/NFAT (35, 63). In PASMC, we have shown that ET-1-induced NFATc3 nuclear import requires elevated [Ca2⫹]i, increased RhoA/ ROCK activity, and a dynamic actin cytoskeleton (24). In addition, NFATc3 nuclear export is regulated by phosphory-

% F340/F380 baseline

NFATc3-EGFP FN/FC

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Fig. 8. O2 does not affect [Ca ]i. Peak and plateau fura 2 F340/F380 values from baseline in human PASMC treated with tempol (3 mM) for 30 min followed by ET-1 (100 nM) are shown. Values are means ⫾ SE; n ⫽ 3. Statistical significance was tested by Student’s t-test.

AJP-Cell Physiol • doi:10.1152/ajpcell.00244.2014 • www.ajpcell.org

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O2·⫺ AND H2O2 HAVE OPPOSITE EFFECTS ON NFATc3

F/G Actin (Intensity)

0.8 0.6 0.4

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A NFATc2 mRNA (2-ΔΔCT)

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NFATc3 mRNA (2-ΔΔCT)

H2O2 on NFATc3 nuclear import were not mediated by JNK1/2. Our data show that degradation of H2O2 increases PASMC [Ca2⫹]i. This increase in [Ca2⫹]i was required for NFATc3 nuclear import and activation. Our results suggest that Ca2⫹ influx through L-type voltage-gated Ca2⫹ channels is partially required, while Ca2⫹ influx through ASIC1 channels is absolutely required, for catalase-induced NFATc3 nuclear import. This is consistent with proposed mechanisms of increased PASMC [Ca2⫹]i by hypoxia, in which hypoxia decreases H2O2, reducing the redox state and, thereby, causing membrane depolarization-mediated Ca2⫹ influx by closing Kv1.5 channels (4, 5). In addition, we recently showed that H2O2 inhibits and PEG-catalase augments store-operated Ca2⫹ entry through ASIC1 in PASMC (59). This is in contrast to studies showing that exogenous H2O2 increases Ca2⫹ release from the endoplasmic reticulum in rat PASMC (27, 47, 48, 71). Our findings are also consistent with the evidence that H2O2 is a vasodilator (75) but contrary to reports that H2O2 induces pulmonary arterial contraction (60). Furthermore, we demonstrate that ET-1-induced increases in O2·⫺ levels in PASMC are not required for the ET-1-induced Ca2⫹ response. Our study also shows that calcineurin activation is necessary for low-H2O2-induced NFATc3 nuclear import and activation. This is consistent with reports showing that exogenous H2O2 inactivates calcineurin (65) in immune cells and in vitro and with results from our study in which H2O2 prevented ET-1induced NFATc3 nuclear import and activation. Regarding a role for the actin cytoskeleton in our findings, our data show that ET-1-induced increases in O2·⫺ levels are implicated in the enhanced actin polymerization caused by ET-1. This is consistent with reports showing that O2·⫺ increases actin polymerization in endothelial cells (23, 53). In addition, it is well established that, in conditions in which O2·⫺ is increased, the activity of RhoA and ROCK, the upstream mediators of actin polymerization, is elevated (4, 14, 40, 45). Although it has been shown that H2O2 activates RhoA in PASMC (20), our data suggest that a reduction in H2O2 might not affect the RhoA/ROCK/actin cytoskeleton pathway, because catalase did not change the ratio of F- to G-actin and did not alter ET-1-induced actin polymerization. We have additionally explored whether changes in NFATc2 or NFATc3 expression could be part of the mechanism of ET-1- or catalase-induced NFAT activation (increased luciferase activity) in pulmonary arteries. It has been shown that

2.5 2.0

ET Catalase Catalase+ET

1.5 1.0 0.5 0.0

Fig. 10. Neither ET-1 nor catalase affects NFATc2 or NFATc3 mRNA levels in pulmonary arteries. Isolated pulmonary arteries from NFAT-luc mice were incubated for 24 h with ET-1 (100 nM), PEG-catalase (250 U/ml), or ET-1 and PEG-catalase. NFATc2 and NFATc3 mRNA levels were determined by real-time PCR, and ␤-actin was used as the endogenous control. Values are means ⫾ SE; n ⫽ 8 arteries from 8 mice. Statistical significance was tested by 2-way ANOVA followed by Student-Newman-Keuls method.

NFATc2 expression is regulated by the STAT3/Pim1 pathway in PAH PASMC, which, in part, regulates NFATc2 activity (12, 21, 22, 52, 57, 67). However, this pathway has not been explored for NFATc3. We found that neither ET-1 nor catalase or their combination affected NFATc2 or NFATc3 mRNA expression under our experimental conditions. These results are consistent with our recent report showing that NFATc2 expression and activity are not affected in pulmonary arteries of SOD1 KO mice in which NFATc3 is activated (62). Furthermore, these results suggest that upregulation of NFATc2 or NFATc3 expression is not involved in the enhanced NFAT transcriptional activity observed in pulmonary arteries in which O2·⫺ levels were increased and H2O2 levels were decreased. In summary, this study describes the novel finding that ET-1 inhibits SOD1 activity, causing a decrease in H2O2 production and contributing to increases in O2·⫺ levels in PASMC. It also demonstrates that a reciprocal change in O2·⫺ and H2O2 is required for ET-1-induced NFATc3 activation. This reciprocal change is indeed sufficient to activate the transcription factor in PASMC. The mechanism involves O2·⫺-mediated actin polymerization and reduced H2O2-mediated increases in Ca2⫹ influx through ASIC1. GRANTS

0.2 0.0

Fig. 9. O2·⫺ is required for ET-induced increases in cytoskeletal actin polymerization. Ratio of filamentous (F)- to globular (G)-actin was determined in cultured human PASMC treated with ET-1 (100 nM, 5 min) in the presence or absence of tempol (3 mM), PEG-SOD (120 U/ml), or PEG-catalase (250 U/ml). Values are means ⫾ SE; n ⫽ 3– 6. *P ⬍ 0.05 vs. control, #P ⬍ 0.05 vs. ET-1 (by ANOVA followed by Student-Newman-Keuls method).

This work was supported in part by National Institutes of Health Grants R01 HL-088151 (to L. V. Gonzalez-Bosc), R01 HL-111084 (to N. L. Jernigan), and P30 GM-103400 (to J. M. Weaver) and a Clinical and Translational Science Center Pilot Award from the University of New Mexico Health Sciences Center. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors.

AJP-Cell Physiol • doi:10.1152/ajpcell.00244.2014 • www.ajpcell.org

O2·⫺ AND H2O2 HAVE OPPOSITE EFFECTS ON NFATc3 AUTHOR CONTRIBUTIONS J.M.R.-D., N.L.J., and L.V.G.B. are responsible for conception and design of the research; J.M.R.-D., W.G., J.M.W., and L.V.G.B. performed the experiments; J.M.R.-D., W.G., J.M.W., and L.V.G.B. analyzed the data; J.M.R.-D., J.M.W., and L.V.G.B. interpreted the results of the experiments; J.M.R.-D. and L.V.G.B. prepared the figures; J.M.R.-D. and L.V.G.B. drafted the manuscript; J.M.R.-D., W.G., J.M.W., N.L.J., and L.V.G.B. edited and revised the manuscript; J.M.R.-D., W.G., J.M.W., N.L.J., and L.V.G.B. approved the final version of the manuscript. REFERENCES 1. Aguirre JI, Morrell NW, Long L, Clift P, Upton PD, Polak JM, Wilkins MR. Vascular remodeling and ET-1 expression in rat strains with different responses to chronic hypoxia. Am J Physiol Lung Cell Mol Physiol 278: L981–L987, 2000. 2. Ahmed MN, Zhang Y, Codipilly C, Zaghloul N, Patel D, Wolin M, Miller EJ. Extracellular SOD overexpression can reverse the course of hypoxia-induced pulmonary hypertension. Mol Med 18: 38 –46, 2012. 3. Albertoni Borghese MF, Bettini LM, Nitta CH, De Frutos S, Majowicz M, Gonzalez Bosc LV. Aquaporin-2 promoter is synergistically regulated by nitric oxide and nuclear factor of activated T cells. Nephron Extra 1: 124 –138, 2011. 4. Archer SL, Marsboom G, Kim GH, Zhang HJ, Toth PT, Svensson EC, Dyck JR, Gomberg-Maitland M, Thebaud B, Husain AN, Cipriani N, Rehman J. Epigenetic attenuation of mitochondrial superoxide dismutase 2 in pulmonary arterial hypertension. Circulation 121: 2661–2671, 2010. 5. Archer SL, Wu XC, Thebaud B, Nsair A, Bonnet S, Tyrrell B, McMurtry MS, Hashimoto K, Harry G, Michelakis ED. Preferential expression and function of voltage-gated, O2-sensitive K⫹ channels in resistance pulmonary arteries explains regional heterogeneity in hypoxic pulmonary vasoconstriction: ionic diversity in smooth muscle cells. Circ Res 95: 308 –318, 2004. 6. Ardanaz N, Pagano PJ. Hydrogen peroxide as a paracrine vascular mediator: regulation and signaling leading to dysfunction. Exp Biol Med (Maywood) 231: 237–251, 2006. 7. Beauchamp C, Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44: 276 –287, 1971. 8. Beiqing L, Chen M, Whisler RL. Sublethal levels of oxidative stress stimulate transcriptional activation of c-jun and suppress IL-2 promoter activation in Jurkat T cells. J Immunol 157: 160 –169, 1996. 9. Bialecki RA, Stinson-Fisher C, Murdoch W, Bertelsen D, Desiato M, Rumsey W. A novel orally active endothelin-A receptor antagonist, ZD1611, prevents chronic hypoxia-induced pulmonary hypertension in the rat. Chest 114: 91S, 1998. 10. Bierer R, Nitta CH, Friedman JK, Codianni SJ, De Frutos S, Dominguez-Bautista JA, Howard TA, Resta TC, Gonzalez Bosc LV. NFATc3 is required for chronic hypoxia-induced pulmonary hypertension in adult and neonatal mice. Am J Physiol Lung Cell Mol Physiol 301: L872–L880, 2011. 11. Bilan DS, Pase L, Joosen L, Gorokhovatsky AY, Ermakova YG, Gadella TWJ, Grabher C, Schultz C, Lukyanov S, Belousov VV. HyPer-3: a genetically encoded H2O2 probe with improved performance for ratiometric and fluorescence lifetime imaging. ACS Chem Biol 8: 535–542, 2012. 12. Bonnet S, Rochefort G, Sutendra G, Archer SL, Haromy A, Webster L, Hashimoto K, Bonnet SN, Michelakis ED. The nuclear factor of activated T cells in pulmonary arterial hypertension can be therapeutically targeted. Proc Natl Acad Sci USA 104: 11418 –11423, 2007. 13. Braz JC, Bueno OF, Liang Q, Wilkins BJ, Dai YS, Parsons S, Braunwart J, Glascock BJ, Klevitsky R, Kimball TF, Hewett TE, Molkentin JD. Targeted inhibition of p38 MAPK promotes hypertrophic cardiomyopathy through upregulation of calcineurin-NFAT signaling. J Clin Invest 111: 1475–1486, 2003. 14. Broughton BR, Jernigan NL, Norton CE, Walker BR, Resta TC. Chronic hypoxia augments depolarization-induced Ca2⫹ sensitization in pulmonary vascular smooth muscle through superoxide-dependent stimulation of RhoA. Am J Physiol Lung Cell Mol Physiol 298: L232–L242, 2010. 15. Callera GE, Tostes RC, Yogi A, Montezano AC, Touyz RM. Endothelin-1-induced oxidative stress in DOCA-salt hypertension involves NADPH-oxidase-independent mechanisms. Clin Sci (Lond) 110: 243–253, 2006.

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